Erythropoietin (EPO), a 34-kDa glycoprotein hormone, is the major regulator of mammalian erythropoiesis (Krantz, S. B. (1991) Blood 77, 419-434). EPO acts on erythroid progenitor cells by preventing apoptosis (Koury et al. (1990) Science 248, 378-381; Zhuang et al. (1995) J Biol Chem 270, 14500-14504), stimulating proliferation of erythroid precursor cells and by inducing differentiation into mature erythrocytes. These effects are transduced by binding of EPO to a specific erythropoietin receptor (EPO-R) on the surface of committed erythroid progenitor cells (Youssoufian et al. (1993) Blood 81, 2223-2236). Deletion of EPO or EPO-R genes in mice has shown that EPO is crucial for the survival, proliferation and differentiation of late committed progenitors (colony forming unit-erythroid, CFU-E), but not of early progenitors (burst forming erythroid, BFU-E) (Wu et al. (1995) Cell 83, 59-67). Mice homozygous for a deletion of either EPO or EPO-R genes die during embryogenesis due to failure of erythropoiesis in the fetal liver.
The EPO-R is a member of the cytokine receptor type I superfamily, which includes the receptors for interleukins (IL) 2-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-stimulating factor (G-CSF), growth hormone (GH), prolactin, thrombopoietin (TPO), leukemia inhibitory factor (LIF), and leptin (Bazan, J. F. (1990) Proc. Natl. Acad. Sci. 87, 6934-6938; Alexander et al. (1995) EMBO J. 14, 5569-5578; Tartaglia et al. (1995) Cell 83, 1263-1271).
Evidence for EPO induced receptor dimerization is based primarily on constitutively active EPO-R mutants, which contain point mutations introducing cysteine substitutions into the extracellular domain at amino acid positions R129, E132, and E133 (Longmore et al. (1991) Cell 167, 1089-102; Yoshimura et al. (1990) Nature 348, 647-649; Watowich et al. (1992) Proc Natl Acad Sci USA 89, 2140-2144; Watowich et al. (1994) Mol Cell Biol 14, 3535-3549; Longmore et al. (1994) Mol Cell Biol 14, 2267-2277). The EPO-R mutants form disulfide-linked homodimers in the endoplasmatic reticulum and on the cell surface (Watowich et al. (1992)). Based on sequence alignments with the related GH receptor, these mutations are expected to be in the receptor-dimer interface region. Expression of the constitutively active EPO-R (R129C) mutant in BaF3 cells results in factor-independent proliferation, and expression in primary cultures of mouse fetal liver cells induce EPO-independent erythroid differentiation (Pharr et al. (1993) Proc Natl Acad Sci USA 90, 938-942). Furthermore, mice infected with a retrovirus carrying the EPO-R (R129C) mutant develop erythroleukemia (Longmore et al. (1994)). Truncated signal transduction inactive receptor mutants lacking part of the intracellular signaling domain are dominant-negative when coexpressed with wild-type EPO-R (Watowich et al. (1994); Barber et al. (1994) Mol Cell. Biol 14, 2257-2265). Both wild-type and truncated receptors can be coimmunoprecipitated with an antibody directed against the C-terminus of the wild-type receptor, which is not present in the truncated form (Miura et al. (1993) Arch Biochem Biophys 306, 200-208), further suggesting the presence of receptor dimers on the cell surface.
Although dimerization of EPO-R is required, it is not sufficient for complete activation of cells. Other accessory cellular factors may be required to send a proliferation signal and, furthermore, these factors may be different from those required to send a differentiation signal. It is desirable to identify molecules other than EPO that activate the EPO-R and stimulate erythropoiesis and this invention meets that need.